Rhizobacteria are plant-associated bacteria, derived from many genera, that have the ability to colonize roots aggressively (Schroth and Hancock, Ann. Rev. Microbiol. 35:453-576 (1981)). Plant growth-promoting rhizobacteria (PGPR) also are able to improve plant growth either through direct effects on the plant (Lugtenberg et al., Curr. Opin. Biotechnol. 2:457-464 (1991)) or by suppressing soilborne plant pathogens (O'Sullivan and O'Gara, Microbiol. Rev. 56:662-676 (1992); Weller, Ann. Rev. Phytopathol. 26:379-407 (1988)). PGPR when applied to soil, seeds or seed pieces, colonize the surface or inside of roots and channels in the rhizosphere that allow physical access to the root. Thus, they are ideally positioned to limit the establishment or spread of pathogens on the roots.
Bacteria introduced for the purpose of suppressing soilborne plant pathogens may interact with pathogens directly through one or more mechanisms of antagonism including competition, parasitism and predation, and antibiosis, or they may function indirectly to limit the initiation or spread of disease by triggering systemic defense responses in the host plant. It has become increasingly clear over the past decade that antibiosis, the inhibition or destruction of one organism by a metabolic product of another, has a dominant role in the control of several important fungal root and seed pathogens by bacterial biocontrol agents, and especially by fluorescent Pseudomonas species (Weller and Thomashow, pp. 173-180 in Pest Management: Biologically Based Technologies, R. D. Lumsden and J. L. Vaughn, eds. (1993)).
Pseudomonas is one of comparatively few bacterial genera capable of synthesizing an array of compounds with broad-spectrum antibiotic activity, and many of the most efficient bacterial biocontrol agents are fluorescent Pseudomonas strains. Antibiotics produced by strains of fluorescent Pseudomonas spp. with biocontrol activity include pyoluteorin and pyrrolnitrin, implicated in control of damping-off diseases of cotton caused by Pythium ultimum and Rhizoctonia solani (Howell et al., Phytopathology 69:480-482 (1979) and Phytopathology 70:712-715 (1980)); oomycin A, also involved in suppression of damping-off of cotton caused by P. ultimum (Gutterson, Crit. Rev. Biotechnol. 10:69-91 (1990)); 2,4-diacetylphloroglucinol, involved in control of take-all disease of wheat caused by Gaeumannomyces graminis var. tritici (Harrison et al., Soil Biol. Biochem. 25:215-221 (1993); Keel et al., Mol. Plant-Microbe Interact. 5:4-13 (1992); Vincent et al., Appl. Environ. Microbiol. 57:2928-2934 (1991)); and phenazine-1-carboxylic acid and its derivatives, active in suppression of take-all (Thomashow et al., J. Bacteriol. 170:3499-3508 (1988); Pierson et al., Mol. Plant-Microbe Interact. 5:330-339 (1992)). Of these pathogens, Gaeumannomyces graminis and Rhizoctonia species are particularly problematic because there are no satisfactory seed treatments for their control. Rhizoctonia and Pythium species are important because they can infect and causing damping-off and root rot diseases in a wide variety of crop plants. For these reasons, biocontrol agents active against these pathogens are of substantial interest to agriculture.
Whereas most individual biocontrol agents function acceptably only within fairly limited circumstances, biologically active plant-associated microorganisms in the aggregate have almost unlimited genetic biodiversity and are adapted to a wide range of environments. Thus, biocontrol agents that can both antagonize plant pathogens and compete successfully with the indigenous rhizosphere microflora of diverse crops or agroecosystems, are desirable. One proposed approach to obtain such biocontrol agents is to identify genetic elements that can confer or enhance the antifungal activities of rhizosphere colonists indigenous to and highly competitive in the plant and ecological environments where biological control is needed, and use these elements to genetically engineer strains of biocontrol agents. It is preferred that the biocontrol agents combine the ability to control the growth of one or more fungal pathogens with other desirable attributes such as adaptation to a particular host plant or environment or the ability to rapidly achieve peak growth rates.
The antibiotic 2,4-diacetylphloroglucinol (Phl) is a phenolic compound of possible polyketide origin with antifungal, antibacterial, antiviral, antihelminthic and phytotoxic properties. Phl is produced by fluorescent pseudomonads that suppress root diseases caused by a variety of soilborne plant pathogens of crops around the world. These include root rot of wheat caused by Fusarium oxysporum, black root rot of tobacco caused by Thielaviopsis basicola (Keel et al., Symbiosis 9:327-341 (1990); damping-off of sugar beet caused by Pythium ultimum (Fenton et al., Appl. Environ. Microbiol. 58:3873-3878 (1992)); damping-off of cotton caused by P. ultimum and Rhizoctonia solani (Kraus et al., Phytopathology 82:264-271 (1992)), blotch of wheat caused by Septoria tritici (Levy et al., Plant Pathol. 41:335-341 (1992)), and take-all of wheat caused by Gaeumannomyces graminis (Harrison et al., supra; Keel et al., (1992) supra; Vincent et al., supra). Strains that produce Phl therefore have considerable agricultural significance.
Three classes of DNA clones have been reported to affect Phl production. The first class contains genes including gacA (Laville et al., Proc. Natl. Acad. Sci. USA 89:1562-1566 (1992)), lemA (PCT Application WO 94/01561; Corbell et al., Mol. Ecol. 3:608 (1994)) and rpoS (Sarniguet et al., Mol. Ecol. 3:607 (1994)), the products of which function as global regulators of a variety of secondary metabolic pathways including that for the synthesis of Phl, thereby indirectly influencing Phl production.
The second class includes DNA sequences of unknown function encoded on the plasmids pME3128 (Keel et al., 1992, supra) and pME3090 (Maurhofer et al., Phytopathology 82:190-195 (1992)) from strain CHA0. The former complemented the Tn5 Phl.sup.- mutant CHA625 to Phl.sup.+ and the latter was selected for its ability to cause overproduction of pyoluteorin when introduced into wild-type CHA0; it subsequently was found also to increase Phl production by about 50%. Neither of these loci has been implicated directly in Phl synthesis, nor reported to be able to confer Phl production to strains deficient in this capacity.
A third class of DNA sequences known to influence Phl production includes those reported by Vincent et al., supra; Fenton et al., supra, and Hara, et al. (Hara et al., pp. 247-249 in Improving Plant Productivity with Rhizobacteria, Ryder, Stephens and Bowen, eds. (1994)) that are capable of transferring Phi biosynthetic capability. Vincent et al., supra, described a locus from P. fluorescens (formerly aureofaciens) Q2-87 (Pierson and Weller, Phytopathology 84:940-947 (1994)) that, when disrupted with the transposon Tn5, resulted in the mutant Q2-87::Tn5-1, which was unable to synthesize Phl. Either of two cosmid clones designated pMON5117 and pMON5118 and isolated from genomic DNA of strain Q2-87 restored antifungal activity and Phl production to Q2-87::Tn5-l.
Mobilization of pMON5118 into two Phl-nonproducing strains conferred the ability to synthesize Phl and increased their antagonistic activity in vitro against Gaeumannomyces graminis, Pythium ultimum, and Rhizoctonia solani. Vincent et al. did not provide any information as to whether a particular portion of the cloned fragment was required, or if the transferred sequences functioned indirectly as a global regulator or specifically to encode enzymes that catalyze the synthesis of Phl.
Fenton et al., supra, reported that pCU203, containing a 6-kb fragment of DNA cloned from P. fluorescens F1 13, partially restored Phl production to a Phl.sup.- Tn5 mutant of F113 and transferred Phl biosynthetic capability only to Ml 14, one of eight nonproducer strains into which it was introduced. Strains F113(pCU203) and M114(pCU203) were more inhibitory to P. ultimum in vitro and increased sugarbeet seedling emergence in soil relative to the parental strains. The 6-kb fragment carried monoacetylphloroglucinol transacetylase activity (Shanahan et al., Anal. Chem. 272:271-277 (1993)). Fenton et al. did not indicate that a particular portion of the cloned fragment was required, or if the transferred sequences functioned indirectly as a global regulator or specifically to encode enzymes that catalyze the synthesis of Phl. Shanahan et al. likewise did not specify what portion of the 6-kb fragment was required for the transacetylase reaction, nor did they indicate or suggest that the fragment contains genetic information sufficient to encode the full complement of enzymes required to catalyze Phl biosynthesis. Neither Shanahan et al. nor Fenton et al. have demonstrated that the transacetylase activity is required for or participates in the Phl biosynthetic pathway in F113.
Hara et al., supra, reported that all of eight strains of Phl-nonproducing strains of fluorescent Pseudomonas spp., when transformed with the plasmid pPHL5122 containing a 7-kb fragment of DNA from Q2-87, produced Phl, and that the overall severity of take-all was reduced on seedlings of wheat treated with strains that contained the cloned Phl locus as compared to those treated with unmodified parental strains. Hara et al. did not indicate whether a particular portion of the cloned fragment was required, or suggest any particular biological function, e.g., catalytic or regulatory, for the transferred sequences.